1. Field of the Invention
The invention relates to a reagent for classification and counting immature cells contained in a liquid sample such as blood.
2. Description of the Related Art
Various blood cells such as erythrocytes, leukocytes and platelets are included in peripheral blood of normal subject. The blood cells are produced in a bone marrow and transferred to a blood stream, while growing in accordance with differentiating themselves from immature cells.
For example, leukocytes such as neutrophils, eosinophils and basophils are differentiated from immature cells to mature cell through (myeloblast.fwdarw.promyelocyte.fwdarw.myelocyte.fwdarw.metamyelocyte) to (stab cell.fwdarw.segmented cell). In a peripheral blood collected from a normal subject, immature cells such as myeloblasts, promyelocytes, myelocyte and metamyelocyte do not appear and stab cells are a small number. However, immature leukocytes appear in some specific cases of, for example, bloods collected from patients suffering blood diseases such as leukemia, metastasis of cancer to bone marrow and severe infectious disease. Thus, it is important and significant to measure immature leukocytes for diagnosis of such diseases.
As one technique for an automated classification and counting of blood cells, there is known a method of recording images of cells and processing. In other case, blood cells are automatically classified and counted by passing them suspended in a diluent through an aperture and processing signals obtained from the respective corpuscles. Lately, the latter's flow system is preferably used in view of accuracy, cost or the like.
According to the flow system, blood cells are suspended in a diluent, and detected by signals based on the respective cells, for example, by signals based on the difference in an optical property and an electric property. That is, they may be detected by using a flow cytometry for detecting scattered light or fluorescent light based on the difference in an optical property, or by using a blood cell counter for detecting electric signals generated from blood cells when the blood cells pass through an aperture therein to which is applied an electric current based on the difference in an electric property. The latter can be further classified into either a DC method for applying direct current to detect signals based on the difference in electric resistance of blood cells and an RF method for applying high frequency current in several MHz to detect signals based on the difference in dielectric constant of blood cells. The DC method detects signals sized in proportion to the volume of the cells, while the RF method detects signals reflecting information on internal structure (the size of nucleus) and constituting substances of the cells.
For example, (1) WO88/09504 and (2) European Patent Application No. 044240 Al describe a combination of the DC method and the RF method for classification and counting 5 types of leukocytes and abnormal cells.
The above quoted reference (1) describes the classification and counting of 5 types of normal (mature) leukocytes i.e., lymphocytes, monocytes, neutrophils, eosinophils and basophils, and abnormal cells using polyoxyethylene-based nonionic surfactant or polyoxyethylene-based anionic surfactant. For example, FIG. 18 illustrates the distribution of lymphoblasts n, myeloblasts l, other immature granulocytes k and left shifted distribution j, where it seems that the left shift means the increase of neutrophils showing fewer nuclear segmentation (stab cell neutrophils).
The reference (2) describes the classification and counting of 5 types of leukocytes and other abnormal cells using polyoxyethylene-based nonionic surfactant under an acidic and hypotonic conditions. FIG. 5 illustrates the distribution of abnormal cells e such as leukemic cells.
In the meanwhile, a variety of diluents and preservative solutions for blood are also known. For example, there are known preservative solutions containing amino acids, which act to adsorb on outer membrane of cell and maintain its morphology (cell protection).
The references (1) and (2) have the drawbacks that there remain unclassified immature cells and there is insufficient accuracy of classification because they primarily aim to classify mature leukocytes into five types and immature cells are additionally classified and counted. For example, when only a polyoxyethylene-based nonionic surfactant is used, mature leukocytes and immature leukocytes are not clearly differentiated because of insufficient shrinkage of mature leukocytes and insufficiently lysing of erythrocytes.